RNA silencing is an innate antiviral defense mechanism in plants that works primarily to decrease expression of viral proteins through RNA degradation. In an effort to overcome RNA silencing, plant viruses encode RNA silencing suppressor proteins. In recent years, similar RNA silencing phenomena have been observed in mammalian cells and viruses. The US11 protein of herpes simplex virus type l (HSV-1) is thought to have silencing suppressor activity in mammalian cells and to help the virus replicate more efficiently. Recent results demonstrate that HSV-1 infection suppresses transient RNA silencing starting very early after infection. It was shown that the US11 virion tegument protein, but not 3 immediate-early proteins, could suppress silencing in plants and in mammalian cells, independent of other HSV-1 genes. Based on the hypothesis that US11 suppresses RNA silencing by interacting with specific proteins or RNAs, the following specific aims are proposed to gain a better understanding of the mechanism of action of US11: 1) Minimum domains of US11 required to suppress silencing will be mapped. The importance in silencing suppression of US11 domains will be determined by making truncated mutants or fusion proteins containing US11 domains and testing the mutants in plant and mammalian assay systems;2) Physical interactions of wild-type and mutant US11 proteins that have little or no silencing suppressor activity will be examined with known components of the RNA-induced silencing complex (RISC), including TRBP (a PACT homolog), Argonaute 2 (Ago2), and Dicer. Protein interactions within cells will be determined by co-precipitation of transiently expressed, epitope-tagged proteins. The selective ability of wild-type and mutant forms of US11 to bind to primary miRNAs, pre-miRNAs, siRNAs, or fully processed miRNAs will be assessed;3) Mechanism(s) by which US11 suppresses silencing will be determined by using in vitro biochemical analysis. Specifically, the effect of US11 on loading of pre-miRNA into RISC-loading complexes (RLCs), the ability of RLCs to process pre-miRNAs to mature miRNAs, and the ability of holo-RISC (RLC+miRNA) to cleave its mRNA target will be determined. PUBLIC HEALTH RELEVANCE: The work proposed in this application will help gain insight into silencing suppression mechanisms and the biological consequences of this viral defense system. A deeper knowledge in this area will ultimately improve public health by aiding in development of unique strategies to interfere with the replication and spread of HSV or other viruses that encode proteins with similar silencing suppressor activities.